Chitosan is a linear copolymer of D-glucosamine (2-amino-2-deoxy-D-glucose) and N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose), obtained by partial (usually >80%) deacetylation of chitin, the main component of exoskeletons of insects and crustaceans. Chitosan has a low oral toxicity (oral LD50: >10,000 mg/kg in mouse and >1500 mg/kg in rats) and has been used as a component in various dietary supplements. In addition, chitosan is safe for topical use and has been used as an ingredient of medical devices or cosmetics. Chitosan is considered to be a safe and biocompatible material, and has been widely explored as a pharmaceutical excipient for a variety of applications such as wound healing, surgical adhesives, mucoadhesive oral drug/gene delivery, gene delivery, and tissue engineering.
Furthermore, chitosan is known to have a pKa of approximately 6.5. Therefore, chitosan is insoluble at a neutral pH but is positively charged and water-soluble at an acidic pH. Although the limited solubility of chitosan at a neutral pH is hypothesized to allow for formation of nanoparticle drug/gene delivery platforms, such limited solubility is disadvantageous for applications of a solution of chitosan at physiological conditions.
In addition, some studies suggest that chitosans exhibit harmful biological effects when administered parenterally. For example, chitosan has been shown in some studies to cause a haemostatic effect and activation of complement following administration to an animal. Moreover, some studies suggest that chitosan induces pro-inflammatory cytokines or chemokines after administration. For example, intraperitoneal (IP) administration of chitosan has been shown to induce a large number of macrophages with hyperplasia in the mesenterium of mice and causes severe peritoneal adhesions in rabbits. In order for nanoparticles to be compatible with parenteral applications, the nanoparticles should not activate immune cells in the bloodstream (monocytes, platelets, leukocytes, and dendritic cells) or in tissues (resident phagocytes) because such activation could cause premature removal of the nanoparticles from the body, and/or elicit inflammatory responses in the body, following administration.
Therefore, there exists a need for chitosan derivatives that can be safely and effectively used as nanoparticles for parenteral administration to animals. Moreover, new and effective methods of utilizing a chitosan derivative, or compositions containing a chitosan derivative, are also very desirable. Accordingly, the present disclosure provides chitosan derivatives and methods of using the chitosan derivatives that exhibit desirable properties and provide related advantages for improvement in safety and efficacy after administration.
Additionally, chitosan derivatives may be advantageously utilized to aid in the delivery of other pharmaceutical compositions, such as dendrimers. Polyamidoamine (“PAMAM”) dendrimers have previously been explored as pharmaceutical compositions for the delivery of therapeutic or imaging agents. PAMAM dendrimers can have various functional groups on their surface, for example amines, carboxylates, and amidoethylethanolamines. In particular, amine-terminated PAMAM dendrimers may be useful for gene delivery to animals because of their cationic charge, which allows for complexation of nucleic acids and for cellular uptake of the dendrimers. Moreover, protonation of tertiary amines in the interior of PAMAM dendrimers may facilitate endosomal escape via a “proton sponge” effect Amine-termini of PAMAM dendrimers may also be useful for covalent conjugation of drugs via linkers cleaved by a condition unique to target tissues.
However, despite their ability to carry various agents, amine-terminated PAMAM dendrimers are generally not useful for systemic applications because of the non-specific toxicity and high risk associated with uptake by the reticuloendothelial system (RES). In an attempt to reduce the charge-related toxicity and prevent opsonization of cationic PAMAM dendrimers, a portion of their amine termini may be modified with polyethylene glycol (PEG), a hydrophilic linear polymer that masks the cationic charge. However, even after modification with polyethylene glycol (i.e., “pegylation”), modified PAMAM dendrimers can be disadvantageous due to the interference of PEG with the target cells. For example, PEG can interfere with important interactions between the carrier and the target cell, causing cellular uptake of the dendrimers to potentially be reduced. Even further modification of the pegylated PAMAM dendrimers with folate, transferrin, or RGD peptide (i.e., ligands known to enhance interactions with target cells) does not solve the problems because the fraction of target cells that express corresponding cellular receptors is not always predictable, and the expression level can change during progression of the disease(s) to be treated.
Therefore, there exists a need for the modification of dendrimers (e.g., PAMAM dendrimers) to provide for their safe and effective administration to animals. Moreover, new and effective methods of utilizing such modified dendrimers are also very desirable. Accordingly, the present disclosure also provides a nanoparticle structure comprising a derivative of chitosan and a dendrimer, as well as methods of using the nanoparticle structure, that exhibit desirable properties and provide related advantages for improvement in administering dendrimers to animals.
The present disclosure provides a polymer comprising a derivative of chitosan, wherein the derivative is zwitterionic, as well as methods of using the polymer. In addition, the present disclosure provides a nanoparticle structure comprising a derivative of chitosan and a dendrimer, as well as methods of utilizing the nanoparticle structure.
The chitosan derivative, the nanoparticle structure, and the methods according to the present disclosure provide several advantages compared to other compositions and methods in the art.
First, the chitosan derivative has a unique pH-dependent charge profile, with isoelectric points (pI) that are tunable among a pH range from about 4 to about 7. The chitosan derivative is zwitterionic and, thus, is soluble in water at pHs below and above the pI, according to the change of its net charge. As a result, the zwitterionic chitosan derivative can be used for parenteral applications, specifically as a component of nanoparticulate drug delivery systems.
Second, the zwitterionic chitosan derivative demonstrates excellent compatibility with blood components and is well tolerated following IP injection. Compared to its precursors (e.g., low molecular weight chitosan), the zwitterionic chitosan derivative has a reduced potential to cause hemolysis, complement activation, and pro-inflammatory response.
Third, the zwitterionic chitosan derivative demonstrates a lower incidence of causing tissue reactions and has a reduced propensity to induce pro-inflammatory cytokine production from macrophages. The zwitterionic chitosan derivative surprisingly suppresses pro-inflammatory responses of activated macrophages.
Fourth, the zwitterionic chitosan derivative can advantageously be used to decrease endotoxins in a composition or in a subject. Endotoxins are a product of the cell wall of gram-negative bacteria and are a common cause of toxic reactions due to their potent stimulation of the mammalian immune system. It is believed that the zwitterionic chitosan derivative may be able to bind to lipopolysaccharides in order to decrease endotoxin levels. Thus, the zwitterionic chitosan derivative may provide an efficient and cost-effective way of removing endotoxin from pharmaceutical products or may serve as a portable reagent for water treatment, such as in war zones or underdeveloped countries.
Fifth, the zwitterionic chitosan derivative can be used to provide safe delivery of cationic polymer nanoparticles to cells by shielding them in normal conditions and activating them only upon exposure to common features of diseased tissues or organs. For example, the microenvironment of a tumor could advantageously be used in this regard. Cancer cells distant from blood vessels are typically deprived of oxygen and undergo anaerobic glycolysis to generate excess lactic acid. As a result, hypoxic tumors tend to develop a weakly acidic microenvironment (e.g., a pH of about 6.5 to about 7.2) compared to normal tissues. Accordingly, the zwitterionioc chitosan derivative of the present disclosure can be used to modify the surface of an amine-terminated PAMAM dendrimer and shield its cationic surface of the dendrimer allow cellular entry in a pH-responsive manner. In an acidic environment, the zwitterionic chitosan derivative can undergo charge reversal, thus allowing PAMAM to interact with tumor cells. For example, following charge reversal, the dendrimer could be effectively delivered to the cell, an agent carried by the dendrimer could be effectively delivered to the cell, or a combination of both.
The following numbered embodiments are contemplated and are non-limiting:
1. A nanoparticle structure comprising a derivative of chitosan and a dendrimer.
2. The nanoparticle of clause 1, wherein the nanoparticle structure is a complex of the derivative of chitosan and the dendrimer.
3. The nanoparticle of clause 2, wherein the complex is an electrostatic complex.
4. The nanoparticle of any one of clauses 1 to 3, wherein the nanoparticle structure has a ratio of derivative:dendrimer at about 1:1.
5. The nanoparticle of any one of clauses 1 to 3, wherein the nanoparticle structure has a ratio of derivative:dendrimer at about 2:1.
6. The nanoparticle of any one of clauses 1 to 3, wherein the nanoparticle structure has a ratio of derivative:dendrimer at about 3:1.
7. The nanoparticle of any one of clauses 1 to 3, wherein the nanoparticle structure has a ratio of derivative:dendrimer at about 4:1.
8. The nanoparticle of any one of clauses 1 to 7, wherein the nanoparticle structure has a critical association concentration between about 2.0 μg/mL and about 3.0 μg/mL.
9. The nanoparticle of any one of clauses 1 to 8, wherein the nanoparticle structure has a critical association concentration of about 2.5 μg/mL.
10. The nanoparticle of any one of clauses 1 to 8, wherein the nanoparticle structure has a critical association concentration of about 2.7 μg/mL.
11. The nanoparticle of any one of clauses 1 to 10, wherein the size of the nanoparticle structure is between about 100 nm and about 500 nm.
12. The nanoparticle of any one of clauses 1 to 10, wherein the size of the nanoparticle structure is between about 200 nm and about 400 nm.
13. The nanoparticle of any one of clauses 1 to 12, wherein the size of the nanoparticle structure is about 200 nm.
14. The nanoparticle of any one of clauses 1 to 12, wherein the size of the nanoparticle structure is about 250 nm.
15. The nanoparticle of any one of clauses 1 to 12, wherein the size of the nanoparticle structure is about 300 nm.
16. The nanoparticle of any one of clauses 1 to 12, wherein the size of the nanoparticle structure is about 350 nm.
17. The nanoparticle of any one of clauses 1 to 12, wherein the size of the nanoparticle structure is about 400 nm.
18. The nanoparticle of any one of clauses 1 to 17, wherein the derivative is zwitterionic.
19. The nanoparticle of any one of clauses 1 to 18, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
20. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 4.5.
21. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 5.0.
22. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 5.5.
23. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 6.0.
24. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 6.5.
25. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 6.8.
26. The nanoparticle of any one of clauses 1 to 19, wherein the derivative has a pI of about 7.0.
27. The nanoparticle of any one of clauses 1 to 26, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
28. The nanoparticle of any one of clauses 1 to 27, wherein the derivative has an An/Am ratio of about 0.3.
29. The nanoparticle of any one of clauses 1 to 27, wherein the derivative has an An/Am ratio of about 0.4.
30. The nanoparticle of any one of clauses 1 to 27, wherein the derivative has an An/Am ratio of about 0.5.
31. The nanoparticle of any one of clauses 1 to 27, wherein the derivative has an An/Am ratio of about 0.6.
32. The nanoparticle of any one of clauses 1 to 27, wherein the derivative has an An/Am ratio of about 0.7.
33. The nanoparticle of any one of clauses 1 to 32, wherein the dendrimer is poly(amidoamine) (“PAMAM”).
34. The nanoparticle of clause 33, wherein the PAMAM dendrimer is an amine-terminated G5 PAMAM dendrimer.
35. A method of delivering a dendrimer to a cell, said method comprising the step of administering a nanoparticle structure comprising a derivative of chitosan and a dendrimer to the cell.
36. The method of clause 35, wherein the cell is a cancer cell.
37. The method of clause 35 or clause 36, wherein the nanoparticle structure releases the dendrimer to the cell.
38. The method of clause 37, wherein the release occurs at an acidic pH.
39. The method of clause 38, wherein the acidic pH is caused by hypoxia.
40. The method of clause 38, wherein the acidic pH is caused by the Warburg effect.
41. The method of any one of clauses 36 to 40, wherein the delivery to the cell is entry into the cell.
42. The method of clause 41, wherein the entry into the cell results in apoptosis of the cell.
43. The method of clause 42, wherein the apoptosis results from delivery of the dendrimer to the cell.
44. The method of clause 42, wherein the apoptosis results from delivery of an agent to the cell and wherein the agent is contained within the dendrimer or covalently conjugated to the dendrimer.
45. A method of delivering a dendrimer to a cell in a subject, said method comprising the step of administering an effective amount of a nanoparticle structure to the subject, wherein the nanoparticle structure comprises a derivative of chitosan and a dendrimer.
46. The method of clause 45, wherein the cell is associated with a tumor in the subject.
47. The method of clause 46, wherein the tumor is a solid tumor.
48. The method of any one of clauses 45 to 47, wherein the nanoparticle structure releases the dendrimer to the cell in the subject.
49. The method of clause 48, wherein the release occurs at an acidic pH.
50. The method of clause 49, wherein the acidic pH is caused by hypoxia.
51. The method of clause 50, wherein the acidic pH is caused by the Warburg effect.
52. The method of any one of clauses 45 to 51, wherein the delivery to the cell is entry into the cell.
53. The method of clause 52, wherein the entry into the cell results in apoptosis of the cell.
54. The method of clause 53, wherein the apoptosis results from delivery of the dendrimer to the cell.
55. The method of clause 54, wherein the apoptosis results from delivery of an agent to the cell and wherein the agent is contained within the dendrimer or covalently conjugated to the dendrimer.
56. A method of delivering an agent to a subject, said method comprising the step of administering a nanoparticle structure to the subject, wherein the nanoparticle structure comprises a derivative of chitosan, a dendrimer, and the agent.
57. The method of clause 56, wherein the agent is contained within the dendrimer or covalently conjugated to the dendrimer.
58. The method of clause 56 or clause 57, wherein the agent is delivered to a cell in the subject.
59. The method of any one of clauses 56 to 58, wherein the agent is a pharmaceutical compound.
60. The method of clause 59, wherein the pharmaceutical compound is an anticancer drug.
61. The method of any one of clauses 56 to 58, wherein the agent is an imaging agent.
62. The method of any one of clauses 56 to 61, wherein the cell is a cancer cell.
63. The method of any one of clauses 56 to 62, wherein the cell is associated with a tumor in the subject.
64. The method of clause 63, wherein the tumor is a solid tumor.
65. The method of any one of clauses 56 to 64, wherein the nanoparticle structure releases the dendrimer to the cell in the subject.
66. The method of clause 65, wherein the release occurs at an acidic pH.
67. The method of clause 66, wherein the acidic pH is caused by hypoxia.
68. A polymer comprising a derivative of chitosan, wherein the derivative is zwitterionic.
69. The polymer of clause 68, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
70. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 4.5.
71. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 5.0.
72. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 5.5.
73. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 6.0.
74. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 6.5.
75. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 6.8.
76. The polymer of clause 68 or clause 69, wherein the derivative has a pI of about 7.0.
77. The polymer of any one of clauses 68 to 76, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
78. The polymer of any one of clauses 68 to 77, wherein the derivative has an An/Am ratio of about 0.3.
79. The polymer of any one of clauses 68 to 77, wherein the derivative has an An/Am ratio of about 0.4.
80. The polymer of any one of clauses 68 to 77, wherein the derivative has an An/Am ratio of about 0.5.
81. The polymer of any one of clauses 68 to 77, wherein the derivative has an An/Am ratio of about 0.6.
82. The polymer of any one of clauses 68 to 77, wherein the derivative has an An/Am ratio of about 0.7.
83. A method of suppressing an inflammatory response in a subject, said method comprising the step of administering an effective amount of a polymer to the subject, wherein the polymer comprises a zwitterionic derivative of chitosan.
84. The method of clause 83, wherein the inflammatory response is associated with activated macrophages in the subject.
85. The method of clause 83 or clause 84, wherein the inflammatory response is pro-inflammatory cytokine production.
86. The method of clause 85, wherein the cytokine production is IL-6 production.
87. The method of clause 85, wherein the cytokine production is TNF-α production.
88. The method of clause 83 or clause 84, wherein the inflammatory response is pro-inflammatory chemokine production.
89. The method of clause 88, wherein the chemokine production is MIP-2 production.
90. The method of any one of clauses 83 to 89, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
91. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 4.5.
92. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 5.0.
93. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 5.5.
94. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 6.0.
95. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 6.5.
96. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 6.8.
97. The method of any one of clauses 83 to 90, wherein the derivative has a pI of about 7.0.
98. The method of any one of clauses 83 to 97, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
99. The method of any one of clauses 83 to 98, wherein the derivative has an An/Am ratio of about 0.3.
100. The method of any one of clauses 83 to 98, wherein the derivative has an An/Am ratio of about 0.4.
101. The method of any one of clauses 83 to 98, wherein the derivative has an An/Am ratio of about 0.5.
102. The method of any one of clauses 83 to 98, wherein the derivative has an An/Am ratio of about 0.6.
103. The method of any one of clauses 83 to 98, wherein the derivative has an An/Am ratio of about 0.7.
104. A method of suppressing cytokine or chemokine production in a subject, said method comprising the step of administering an effective amount of a polymer to the subject, wherein the polymer comprises a zwitterionic derivative of chitosan.
105. The method of clause 104, wherein the cytokine or chemokine production is associated with activated macrophages.
106. The method of clause 104 or clause 105, wherein the cytokine or chemokine production is induced by lipopolysaccharide (LPS).
107. The method of clause 106, wherein the polymer binds directly to the LPS.
108. The method of any one of clauses 104 to 107, wherein the cytokine is IL-6.
109. The method of any one of clauses 104 to 107, wherein the cytokine is TNF-α.
110. The method of any one of clauses 104 to 107, wherein the chemokine is MIP-2.
111. The method of any one of clauses 104 to 110, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
112. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 4.5.
113. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 5.0.
114. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 5.5.
115. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 6.0.
116. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 6.5.
117. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 6.8.
118. The method of any one of clauses 104 to 111, wherein the derivative has a pI of about 7.0.
119. The method of any one of clauses 104 to 118, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
120. The method of any one of clauses 104 to 119, wherein the derivative has an An/Am ratio of about 0.3.
121. The method of any one of clauses 104 to 119, wherein the derivative has an An/Am ratio of about 0.4.
122. The method of any one of clauses 104 to 119, wherein the derivative has an An/Am ratio of about 0.5.
123. The method of any one of clauses 104 to 119, wherein the derivative has an An/Am ratio of about 0.6.
124. The method of any one of clauses 104 to 119, wherein the derivative has an An/Am ratio of about 0.7.
125. A method of binding a lipopolysaccharide, said method comprising the step of contacting the lipopolysaccharide with a polymer comprising a zwitterionic derivative of chitosan.
126. The method of clause 125, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
127. The method of clause 125 or clause 126, wherein the derivative has a pI of about 4.5.
128. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 5.0.
129. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 5.5.
130. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 6.0.
131. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 6.5.
132. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 6.8.
133. The method of any one of clauses 125 to 127, wherein the derivative has a pI of about 7.0.
134. The method of any one of clauses 125 to 133, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
135. The method of any one of clauses 125 to 134, wherein the derivative has an An/Am ratio of about 0.3.
136. The method of any one of clauses 125 to 134, wherein the derivative has an An/Am ratio of about 0.4.
137. The method of any one of clauses 125 to 134, wherein the derivative has an An/Am ratio of about 0.5.
138. The method of any one of clauses 125 to 134, wherein the derivative has an An/Am ratio of about 0.6.
139. The method of any one of clauses 125 to 134, wherein the derivative has an An/Am ratio of about 0.7.
140. A method of decreasing a bacterial toxin in a subject, said method comprising the step of administering an effective amount of a polymer to the subject, wherein the polymer comprises a zwitterionic derivative of chitosan.
141. The method of clause 140, wherein the bacterial toxin is an endotoxin.
142. The method of clause 140 or clause 141, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
143. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 4.5.
144. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 5.0.
145. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 5.5.
146. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 6.0.
147. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 6.5.
148. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 6.8.
149. The method of any one of clauses 140 to 142, wherein the derivative has a pI of about 7.0.
150. The method of any one of clauses 140 to 149, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
151. The method of any one of clauses 140 to 150, wherein the derivative has an An/Am ratio of about 0.3.
152. The method of any one of clauses 140 to 150, wherein the derivative has an An/Am ratio of about 0.4.
153. The method of any one of clauses 140 to 150, wherein the derivative has an An/Am ratio of about 0.5.
154. The method of any one of clauses 140 to 150, wherein the derivative has an An/Am ratio of about 0.6.
155. The method of any one of clauses 140 to 150, wherein the derivative has an An/Am ratio of about 0.7.
156. A method of decreasing a bacterial toxin in a composition, said method comprising the step of administering an effective amount of a polymer to the composition, wherein the polymer comprises a zwitterionic derivative of chitosan.
157. The method of clause 156, wherein the bacterial toxin is an endotoxin.
158. The method of clause 156 or clause 157, wherein the composition is a pharmaceutical composition.
159. The method of clause 156 or clause 157, wherein the composition is water.
160. The method of any one of clauses 156 to 159, wherein the derivative has an isoelectric point (pI) between about 4 and about 7.
161. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 4.5.
162. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 5.0.
163. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 5.5.
164. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 6.0.
165. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 6.5.
166. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 6.8.
167. The method of any one of clauses 156 to 160, wherein the derivative has a pI of about 7.0.
168. The method of any one of clauses 156 to 167, wherein the derivative has an An/Am ratio between 0.3 to 0.7.
169. The method of any one of clauses 156 to 171, wherein the derivative has an An/Am ratio of about 0.3.
170. The method of any one of clauses 156 to 171, wherein the derivative has an An/Am ratio of about 0.4.
171. The method of any one of clauses 156 to 171, wherein the derivative has an An/Am ratio of about 0.5.
172. The method of any one of clauses 156 to 171, wherein the derivative has an An/Am ratio of about 0.6.
173. The method of any one of clauses 156 to 171, wherein the derivative has an An/Am ratio of about 0.7.